Effects of subtherapeutic concentrations of antimicrobials on gene acquisition events in Yersinia, Proteus, Shigella, and Salmonella recipient organisms in isolated ligated intestinal loops of swine

Matt T. Brewer Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011.

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Nalee Xiong Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011.
Dr. Xiong's present address is Department of Neuroscience, University of Minnesota Medical School, Minneapolis, MN 55414.

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Kristi L. Anderson Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011.

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Steve A. Carlson Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011.

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Abstract

Objective—To assess antimicrobial resistance and transfer of virulence genes facilitated by subtherapeutic concentrations of antimicrobials in swine intestines.

Animals—20 anesthetized pigs experimentally inoculated with donor and recipient bacteria.

Procedures—4 recipient pathogenic bacteria (Salmonella enterica serotype Typhimurium, Yersinia enterocolitica, Shigella flexneri, or Proteus mirabilis) were incubated with donor bacteria in the presence of subinhibitory concentrations of 1 of 16 antimicrobials in isolated ligated intestinal loops in swine. Donor Escherichia coli contained transferrable antimicrobial resistance or virulence genes. After coincubations, intestinal contents were removed and assessed for pathogens that acquired new antimicrobial resistance or virulence genes following exposure to the subtherapeutic concentrations of antimicrobials.

Results—3 antimicrobials (apramycin, lincomycin, and neomycin) enhanced transfer of an antimicrobial resistance plasmid from commensal E coli organisms to Yersinia and Proteus organisms, whereas 7 antimicrobials (florfenicol, hygromycin, penicillin G, roxarsone, sulfamethazine, tetracycline, and tylosin) exacerbated transfer of an integron (Salmonella genomic island 1) from Salmonella organisms to Yersinia organisms. Sulfamethazine induced the transfer of Salmonella pathogenicity island 1 from pathogenic to nonpathogenic Salmonella organisms. Six antimicrobials (bacitracin, carbadox, erythromycin, sulfathiazole, tiamulin, and virginiamycin) did not mediate any transfer events. Sulfamethazine was the only antimicrobial implicated in 2 types of transfer events.

Conclusions and Clinical Relevance—10 of 16 antimicrobials at subinhibitory or subtherapeutic concentrations augmented specific antimicrobial resistance or transfer of virulence genes into pathogenic bacteria in isolated intestinal loops in swine. Use of subtherapeutic antimicrobials in animal feed may be associated with unwanted collateral effects.

Abstract

Objective—To assess antimicrobial resistance and transfer of virulence genes facilitated by subtherapeutic concentrations of antimicrobials in swine intestines.

Animals—20 anesthetized pigs experimentally inoculated with donor and recipient bacteria.

Procedures—4 recipient pathogenic bacteria (Salmonella enterica serotype Typhimurium, Yersinia enterocolitica, Shigella flexneri, or Proteus mirabilis) were incubated with donor bacteria in the presence of subinhibitory concentrations of 1 of 16 antimicrobials in isolated ligated intestinal loops in swine. Donor Escherichia coli contained transferrable antimicrobial resistance or virulence genes. After coincubations, intestinal contents were removed and assessed for pathogens that acquired new antimicrobial resistance or virulence genes following exposure to the subtherapeutic concentrations of antimicrobials.

Results—3 antimicrobials (apramycin, lincomycin, and neomycin) enhanced transfer of an antimicrobial resistance plasmid from commensal E coli organisms to Yersinia and Proteus organisms, whereas 7 antimicrobials (florfenicol, hygromycin, penicillin G, roxarsone, sulfamethazine, tetracycline, and tylosin) exacerbated transfer of an integron (Salmonella genomic island 1) from Salmonella organisms to Yersinia organisms. Sulfamethazine induced the transfer of Salmonella pathogenicity island 1 from pathogenic to nonpathogenic Salmonella organisms. Six antimicrobials (bacitracin, carbadox, erythromycin, sulfathiazole, tiamulin, and virginiamycin) did not mediate any transfer events. Sulfamethazine was the only antimicrobial implicated in 2 types of transfer events.

Conclusions and Clinical Relevance—10 of 16 antimicrobials at subinhibitory or subtherapeutic concentrations augmented specific antimicrobial resistance or transfer of virulence genes into pathogenic bacteria in isolated intestinal loops in swine. Use of subtherapeutic antimicrobials in animal feed may be associated with unwanted collateral effects.

Subtherapeutic concentrations of antimicrobials have been used for decades as growth promotants and prophylactic agents. This practice has been scrutinized as a contributor to the dissemination of antimicrobial resistance. Specifically, subtherapeutic concentrations of chlortetracycline have been associated with an increase in resistance to multiple antimicrobials for swine intestinal microbes.1 Another study2 revealed that subtherapeutic concentrations of antimicrobials can lead to the propagation of antimicrobial-resistant Enterococcus organisms in swine. A recent study3 indicated that intestinal bacteriophages are activated by subtherapeutic concentrations of antimicrobials, and this activation can lead to transfer of antimicrobial resistance genes. However, these studies have not addressed specific gene transfer events that precipitate resistance and virulence. Thus, little is known about the effects of these antimicrobials on specific gene transfer events that promote the dissemination of antimicrobial resistance and virulence genes in enteric pathogens.

The objective of the study reported here was to identify antimicrobials that at subtherapeutic concentrations augment the transfer of certain antimicrobial resistance and virulence genes. The main goal was to assess 3 types of transfer events (plasmid transfer, integron transfer, and horizontal transfer of a pathogenicity island) mediated by subtherapeutic concentrations of antimicrobials in vivo. Specifically, the study was conducted to determine the relative rates of antimicrobial-mediated transfer events involving 3 representative antimicrobial resistance plasmids, 1 model integron, and 1 pathogenicity island in the presence of antimicrobials that have been approved for use (currently or in the past) at subtherapeutic concentrations as feed additives for swine.

Materials and Methods

Animals—Twenty juvenile swine were used to assess in vivo transfer events. Pigs were of mixed breeds and both sexes; pigs weighed between 5 and 10 kg. Animal experiments were approved by the Iowa State University Institutional Animal Care and Use Committee.

Pigs were anesthetized with pentobarbitala (40 mg/kg, intraperitoneal). Isolated ligated loops of intestine (9 loops/pig; each ligated loop was 10 cm in length) were created4 with 2-0 silk sutures.b At the end of the experiments, anesthetized pigs were euthanized by intracardiac administration of an overdose of pentobarbital (100 mg/kg).

Plasmid transfer—To determine plasmid transfer from donor bacteria to recipient pathogenic bacteria, each loop was injected with approximately 109 to 1011 CFUs of donor bacteria and 109 CFUs of recipient bacteria in 1 mL of saline (0.9% NaCl) solution that contained 1 of 16 feed additive antimicrobialsc (apramycin, bacitracin, carbadox, erythromycin, florfenicol, hygromycin, lincomycin, neomycin, penicillin G, roxarsone, sulfamethazine, sulfathiazole, tetracycline, tiamulin, tylosin, and virginiamycin); antimicrobial-free saline solution was used as a negative control treatment. Concentration of each antimicrobial was 1 μg/mL. This concentration was chosen because it was less than the established breakpoints for all of the antimicrobial-bacteria combinations,5 and preliminary experiments conducted by our laboratory group on the minimum inhibitory concentration of these antimicrobials revealed that this was the lowest common concentration that permitted growth of all donor and recipient bacteria for each of the 16 antimicrobials (data not shown).

Plasmid transfer from a donor commensal intestinal Escherichia coli6 (antimicrobial susceptible and lacking virulence genes) to each of 4 recipient pathogenic bacteria (Salmonella enterica serotype Typhimurium strain SL1344,7 Yersinia enterocolitica,d Shigella flexneri,e or Proteus mirabilisf) was measured. All of the recipient bacteria were susceptible to most antimicrobials (except tetracycline and streptomycin), as determined by use of microdilution broth assays performed in accordance with standards established by the Clinical and Laboratory Standards Institute.5 The E coli donor strain was experimentally transformed with 1 of 3 conjugative plasmids encoding an ESBL,8 amikacin resistance (aacC4),9 or fluoroquinolone resistance (via the qnr gene).10

Bacteria were allowed to incubate in the isolated ligated intestinal loops for 1 hour, after which loops were excised. Total volume of intestinal content in each isolated loop was 1.1 to 1.2 mL; approximately 10% of the total content of each loop was removed for plating on media selective for each of the 4 pathogens (XLD agarg for Salmonella and Shigella organisms, Yersinia selective agar baseh for Yersinia organisms, and phenylalanine agarg for Proteus organisms); agar contained 1 of 3 antimicrobials (ceftiofur,i 32 μg/mL for the ESBL plasmid; amikacin,c 64 μg/mL; or enrofloxacin,j 8 μg/mL) at their respective breakpoint concentrations.5 The content of each loop was cultured in triplicate (3 agar plates/loop). Control experiments revealed that the donor E coli did not grow on the selective media, except for the XLD agar; however, the E coli colonies that grew on XLD agar were biochemically distinct from the Salmonella and Shigella colonies.

Plates were incubated at 37°C for 16 hours. Bacteria were then enumerated, and the number of translocants/109 recipients was calculated by multiplying the number of recovered colonies by 10 to account for the fact that only 10% of the intestinal contents were plated. The log10 of the product was derived and used for final data analysis and statistical evaluation.

Transfer of the ESBL plasmid was assessed with a PCR assay with primers (5′-ATGATGAAAAAATCGT-TATGCT-3′ and 5′-TTATTGCAGCTTTTCAAGAAT-3′) specific to the blaCMY-2 gene present on the ESBL plasmid.8 Each transfer event was determined for 100 representative colonies.

SGI1 transfer—The possibility that subinhibitory concentrations of antimicrobials can modulate the transfer of SGI1 from Salmonella organisms to Yersinia recipients, Shigella recipients, or Proteus recipients was evaluated. The SGI1 is a multiresistance integron that encodes resistance to 5 antimicrobials in Salmonella organisms.11 Integrons are mobile genomic elements putatively transferred by bacteriophages,12 and studies13–15

have indicated that Yersinia spp, Shigella spp, and Proteus spp are capable of receiving integrons.

To evaluate SGI1 transfer, approximately 109 CFUs of SGI1-free recipient (Yersinia enterocolitica, Shigella flexneri, or Proteus mirabilis [all of which were susceptible to most antimicrobials, except tetracycline and streptomycin, as determined with microdilution broth assays performed in accordance with standards established by the Clinical and Laboratory Standards Institute5]) were each coinoculated with 109 CFUs of SGI1-bearing donor S enterica serotype Typhimurium phagetype DT104 strain LNWI4 into ligated intestinal loops. Coincubations included 1 of the 16 aforementioned antimicrobials (concentration, 1 μg/mL) and the negative control treatment (saline solution without an antimicrobial).

Bacteria were allowed to incubate in the isolated ligated intestinal loops for 1 hour, after which loops were excised. Total volume of the intestinal content in each isolated loop was 1.1 to 1.2 mL; approximately 10% of the total content of each loop was removed for plating on media selective for each of the 3 pathogens (XLD agar for Salmonella and Shigella organisms, Yersinia selective agar base for Yersinia organisms, and phenylalanine agar for Proteus organisms); agar contained 1 of 2 antimicrobials relevant for SGI1 (ampicillin,c 32 μg/mL; chloramphenicol,c 32 μg/mL) at their respective breakpoint concentrations.5 The content of each loop was cultured in triplicate (3 agar plates/loop).

Plates were incubated at 37°C for 16 hours. Bacteria then were enumerated, and the number of translocants/109 recipients was calculated by multiplying the number of recovered colonies by 10 to account for the fact that only 10% of the intestinal contents were plated. The log10 of the product was derived and used for final data analysis and statistical evaluation.

A PCR assay specific to the floR-tetR sequence in SGI116 was used to assess the presence of the SGI1 integron in 100 representative recipient bacteria that grew on selective media containing ampicillin and chloramphenicol. Additionally, transferrants were assessed for the absence of a Salmonella virulence gene segment (sipB/C).16 Control experiments revealed that the donor Salmonella organisms were not able to grow on the media selective for Yersinia spp and P roteus spp, whereas Salmonella organisms were distinguishable from Shigella organisms on XLD agar.

SPI1 transfer—Transfer of SPI1 from pathogenic to nonpathogenic Salmonella organisms was evaluated. The SPI1 is a major determinant of virulence in Salmonella organisms, and avirulent Salmonella organisms lack SPI1.17 The genomic structure of SPI1 suggests that this island is transferrable.18 In vivo coincubations with the 16 antimicrobials and the negative control treatment (saline solution without an antimicrobial) were performed as described previously for the plasmid and integron transfer experiments. The SPI1-bearing S enterica serotype Typhimurium (antimicrobial-susceptible strain SL13447) was incubated with 1 of 4 SPI1-free strains (S enterica serotypes Litchfield,17 Senftenberg,17 Seminole, or Betiocky), all 4 of which were transformed with a nonconjugative plasmid encoding green fluorescent protein.19 For this experiment, 1011 CFUs of donor bacteria and 1011 CFUs of recipient bacteria were used for the incubations.

Bacteria were allowed to incubate in the ligated loops for 1 hour. Total volume of each loop was 1.1 to 1.2 mL; approximately 10% of the total content of each loop was removed and used in a large-volume tissue culture invasion assay20 in which recovered bacteria were plated on XLD agar that contained 50 μg of zeocink/mL (zeocin is the selective marker for the fluorescence plasmid19). The content of each loop was cultured in triplicate (3 agar plates/loop). Fluorescent colonies that were invasive (ie, recovered from inside tissue culture cells) were individually expanded in fresh nutrient broth and then subjected to a second invasion assay. Amount of invasion was compared with that of strain SL1344.7 Serotype analysis was conducted at another laboratory,l and a PCR assay that detected the sipB-sipC sequence in SPI116 was performed on each clone for which invasion was indistinguishable from that of strain SL1344 (approx 1% invasion).

Statistical analysis—Statistical differences were assessed with an ANOVA.m There were 51 combinations of antimicrobials and transfer events ([16 antimicrobials plus 1 antimicrobial-free treatment] times 3 transfer events), each assessed in 3 separate ligated loops (153 total loops) and 3 agar plates/loop (459 total loops). Frequency data for all transfer events were analyzed en masse to allow for interantimicrobial and intraevent comparisons and intra-antimicrobial and interevent comparisons (ie, comparisons were made among the antimicrobials within the 3 transfer events [plasmid, SGI1, and SPI1] and among the 3 transfer events within a specific antimicrobial). The Scheffe F test was chosen as the ad hoc test because our research group has empirically found it to be the most conservative for detecting differences, in contrast to the Bonferroni test. Values of P < 0.05 were considered significant.

Results

Plasmid transfer—In vivo transfer events of 3 clinically relevant antimicrobial resistance plasmids were assessed by use of ligated intestinal loops of swine. Loops were coinoculated with donor commensal E coli bearing 1 of 3 antimicrobial resistance plasmids, 1 of 4 recipient pathogenic Enterobacteriaceae, and an antimicrobial (1 μg/mL) approved as a feed additive in swine. Commensal E coli and recipient Enterobacteriaceae were chosen because these microbes are highly representative of enteric bacteria that transfer genetic information. Three antimicrobials mediated a significant increase in the frequency of a specific plasmid transfer event in vivo (Figure 1). An increased frequency of transfer of the ESBL plasmid from E coli to Yersinia recipients and from E coli to Proteus recipients was evident in the presence of apramycin, lincomycin, or neomycin (Table 1). None of the antimicrobials caused significant changes in the frequencies of ESBL plasmid transfer from E coli to Salmonella recipients and from E coli to Shigella recipients. Additionally, none of the antimicrobials caused significant changes in transfer of amikacin resistance plasmids or fluoroquinolone resistance plasmids.

Figure 1—
Figure 1—

Plasmid transfer from commensal Escherichia coli to Yersinia organisms and Proteus organisms after coincubation with a subtherapeutic concentration (1 μg/mL) of lincomycin (white bars), apramycin (gray bars), or neomycin (striped bars) or with saline (0.9% NaCl) solution that did not contain an antimicrobial (negative control treatment [black bars]) for 1 hour in isolated ligated intestinal loops in swine. Results represent the mean ± SEM for 3 experiments that were each performed in triplicate. Notice that there was enhancement of transfer of the ESBL plasmid8 in the presence of subtherapeutic concentrations of antimicrobials in Yersinia recipients and Proteus recipients. *Within a bacterium, value differs significantly (P < 0.05) from the value for the negative control treatment.

Citation: American Journal of Veterinary Research 74, 8; 10.2460/ajvr.74.8.1078

Table 1—

Gene transfer events for donor bacteria to recipient bacteria after coincubation with a subtherapeutic concentration (1 μg/mL) of 16 antimicrobials or saline (0.9% NaCl) solution that did not contain an antimicrobial (negative control treatment) for 1 hour in isolated ligated intestinal loops in swine.

AntimicrobialESBL plasmid transferSGI1 transferSPI1 transferRatio of total transfer frequencies
Saline solutionNANANA1.0
ApramycinYersinia and Proteus recipients*3,471
Bacitracin2.3
Carbadox0.6
Erythromycin0.7
FlorfenicolYersinia recipients*9.8
HygromycinYersinia recipients*5.1
LincomycinYersinia and Proteus recipients*4,596
NeomycinYersinia and Proteus recipients*4,641
Penicillin GYersinia recipients*4.4
RoxarsoneYersinia recipients*4.7
SulfamethazineYersinia recipients*3 recipient nonpathogenic serovars of Salmonella enterica*13.6
Sulfathiazole1.1
TetracyclineYersinia recipients*4.1
Tiamulin1.2
TylosinYersinia recipients*10.3
Virginiamycin0.9

Mean transfer frequency in the absence of an antimicrobial was approximately 1.4 × 10−8 transconjugates/recipient for the ESBL plasmid, 2.6 × 10−9 translocants/recipient for SGI1, and 3 × 10−11 invasive clones/noninvasive recipient for SPI1; these frequencies were calculated by dividing the total number of all recovered recipients by the total number of donor bacteria added to all of the coincubations in the absence of an antimicrobial. For each antimicrobial, the ratio of total transfer frequencies = (total number of all bacteria that received the new genetic element when coincubated with an antimicrobial/total number of recipients coincubated with an antimicrobial)/(total number of all bacteria that received the new genetic element when coincubated without an antimicrobial/total number of recipients coincubated without an antimicrobial).

Transfer frequency was significantly (P < 0.05) greater than the transfer frequency for the antimicrobial-free (negative control) treatment.

Salmonella enterica serotypes Betiocky, Litchfield, and Seminole.

NA = Not applicable. — = Transfer frequency was not significantly greater than the transfer frequency for the antimicrobial-free (negative control) treatment.

The PCR assay of 100 representative ceftiofur-resistant colonies from each of the aforementioned transfer events revealed that 96% to 97% of the putative transconjugates contained blaCMY-2. Colonies that lacked this sequence were numerically discounted as transconjugates in the final calculations (Figure 1).

SGI1 transfer—In vivo transfer events of an integron were assessed with ligated intestinal loops coinoculated with donor Salmonella organisms bearing SGI1,11 1 of 4 recipient pathogenic Enterobacteriaceae, and an antimicrobial (1 μg/mL) approved as a feed additive in swine. Seven antimicrobials mediated a significant increase in the frequency of in vivo transfer of a specific integron (Figure 2). An increased frequency of transfer of the SGI1 integron from Salmonella organisms to Yersinia organisms was evident in the presence of florfenicol, hygromycin, penicillin G, roxarsone, sulfamethazine, tetracycline, and tylosin (Table 1). None of the antimicrobials caused significant changes in the frequencies of SGI1 transfer events from Salmonella organisms to either of the other 2 pathogens examined. The PCR assay of 100 representative colonies from each of the 16 aforementioned transfer events revealed that 98% to 99% of the putative SGI1 recipients contained the floR-tetR sequence indicative of SGI1.16 Colonies that lacked this sequence were numerically discounted as translocants in the final calculations.

Figure 2—
Figure 2—

Transfer of SGI1 from Salmonella enterica serotype Typhimurium phagetype DT104 to Yersinia enterocolitica after coincubation with a subtherapeutic concentration (1 μg/mL) of 16 antimicrobials or saline solution that did not contain an antimicrobial (negative control treatment) for 1 hour in isolated ligated intestinal loops in swine. Results represent the mean ± SEM for 3 experiments that were each performed in triplicate. Notice that transfer of the integron was enhanced in the presence of 7 antimicrobials. *Value differs significantly (P < 0.05) from the value for coincubation with the negative control treatment. SMZ = Sulfamethazine.

Citation: American Journal of Veterinary Research 74, 8; 10.2460/ajvr.74.8.1078

SPI1 transfer—In vivo transfer events of a pathogenicity island were assessed with ligated intestinal loops coinoculated with donor Salmonella organisms, 1 of 4 recipient nonpathogenic Salmonella organisms that lacked SPI1, and an antimicrobial (1 μg/mL) approved as a feed additive in swine. Sulfamethazine mediated the horizontal transfer of SPI1 in vivo (Figure 3). Three of 4 SPI1-free S enterica serovars (Betiocky, Seminole, and Litchfield) had transfer events in the presence of sulfamethazine (Table 1). Transfer of SPI1 was detected in the absence of an antimicrobial for 1 clone of S enterica serovar Litchfield. Salmonella enterica serovar Senftenberg did not acquire SPI1 in this experiment. Results of the PCR assays confirmed the presence of the sipB-sipC genomic segment in all clones that had invasion indistinguishable from that of strain SL1344.

Figure 3—
Figure 3—

Transfer of SPI1 from pathogenic Salmonella organisms to nonpathogenic Salmonella organisms after coincubation with a subtherapeutic concentration (1 μg/mL) of sulfamethazine (white bars) or saline solution that did not contain an antimicrobial (negative control treatment [black bars]) for 1 hour in isolated ligated intestinal loops in swine. Fluorescent bacteria were subjected to 2 invasion assays, and colonies that were invasive (ie, recovered from inside tissue culture cells) were quantitated by enumerating the bacteria recovered from tissue culture cell lysates at the end of the second assay. Invasion represents the percentage as compared with that of the donor Salmonella organisms. Results represent the mean ± SEM for 3 experiments that were each performed in triplicate. Notice that transfer of SPI1 was only detected in the presence of sulfamethazine and that 1 clone of S enterica serotype Litchfield was invasive in the absence of an antimicrobial. The number above each column indicates the cumulative number of serovar-specific clones that were invasive across 3 separate experiments.

Citation: American Journal of Veterinary Research 74, 8; 10.2460/ajvr.74.8.1078

Discussion

The use of subtherapeutic concentrations of antimicrobials in livestock feed is a controversial practice that is being scrutinized. Of concern are the unknown collateral effects on bacterial gene transcription, plasmid transconjugation from commensals to pathogens, and viral-mediated transduction of genes from commensals to pathogens and from one pathogen to another. Specifically, these collateral effects can activate molecular processes culminating in gene transfer events that yield pathogens with multiple antimicrobial resistance or pathogens with new virulence capabilities.

The experiments in the present study involved the use of a low concentration (1 μg/mL) of antimicrobials to mimic field conditions without directly harming the donor or recipient bacteria. Although this concentration may be lower than concentrations in the intestinal tract of swine fed feed that contains antimicrobials, this concentration was considered relevant for swine in which the antimicrobial is removed from the diet prior to slaughter.

The present study revealed that there were transfer events in the absence of an antimicrobial and that certain antimicrobials mediated gene transfer events at a higher frequency than did other antimicrobials (Table 1). Sulfamethazine mediated 2 separate transfer events (SGI1 transfer and SPI1 transfer), whereas a related drug, sulfathiazole, did not mediate any transfer events. Bacitracin, carbadox, erythromycin, tiamulin, and virginiamycin were also not implicated in transfer events. Transfer events were confirmed with a PCR assay, although a few (1% to 4%) transconjugates and translocants did not harbor the transferrable element, which suggested that efflux systems may have been activated in these few clones.

Three classes of antimicrobials were implicated in transfer of the ESBL plasmid. Lincomycin is a lincosamide, apramycin is an aminocyclitol, and neomycin is an aminoglycoside (although the latter 2 are sometimes grouped in the same class). Lincomycin is an inhibitor of the 50S ribosome in bacteria, yet 3 other 50S inhibitors (erythromycin, tiamulin, and virginiamycin) did not mediate transfer events. Apramycin and neomycin are inhibitors of the 30S ribosome, but another 30S inhibitor, tetracycline, did not exacerbate transfer of the ESBL plasmid. It is possible that apramycin, lincomycin, and neomycin can selectively alter protein synthesis that impacts sex pheromones and conjugation.

Seven antimicrobials (florfenicol, hygromycin, penicillin G, roxarsone, sulfamethazine, tetracycline, and tylosin) from 7 antimicrobial classes were implicated in the transfer of SGI1. Tylosin and florfenicol are 50S inhibitors, but that is the extent of the similarities among these 7 antimicrobials. Because movement of SGI1 may be a phage-mediated event,12 it is possible that those 7 antimicrobials activated phage recrudescense in SGI1-bearing Salmonella organisms, similar to that recently reported for sulfamethazine, chlortetracycline, and penicillin.3 It is also possible that naked SGI1 DNA was transferred from Salmonella organisms to Yersinia organisms, although to our knowledge this process has not been described in the literature.

Analysis of results of the present study indicated that certain antimicrobials at subtherapeutic concentrations are more likely to mediate unwanted gene transfer into pathogenic bacteria in ligated intestinal loops in swine. Sulfamethazine mediated 2 types of transfer, whereas apramycin, lincomycin, and neomycin exerted the greatest quantitative effect on a single transfer event into 2 genera of Enterobacteriaceae. Bacitracin, carbadox, erythromycin, sulfathiazole, tiamulin, and virginiamycin did not significantly influence any of the 3 transfer events evaluated. No antimicrobial class–specific patterns were observed in the 3 transfer events. Transfer of the ESBL plasmid was detected at the highest frequency. Protein synthesis irregularities may underlie transfer of the ESBL plasmid, and activation of bacteriophages may be involved in SGI1 transfer or SPI1 transfer (or both). Regardless of the mechanisms involved, subtherapeutic concentrations of antimicrobials have potential ecologic impacts that involve the dissemination of antimicrobial resistance and virulence genes.

ABBREVIATIONS

ESBL

Extended-spectrum β-lactamase

SGI1

Salmonella genomic island 1

SPI1

Salmonella pathogenicity island 1

XLD

Xylose lysine deoxycholate

a.

Fort Dodge Animal Health, Fort Dodge, Iowa.

b.

Ethicon Inc, Somerville, NJ.

c.

Sigma Chemical Co, St Louis, Mo.

d.

Yersinia enterocolitica 9610, ATCC, Manassas, Va.

e.

Shigella flexneri 9199, ATCC, Manassas, Va.

f.

Proteus mirabilis 4630, ATCC, Manassas, Va.

g.

Becton Dickinson, Franklin Lakes, NJ.

h.

Thermo Fisher Scientific, Pittsburgh, Pa.

i.

Pfizer Inc, Kalamazoo, Mich.

j.

Bayer Corp, Shawnee Mission, Kan.

k.

Invitrogen Corp, Carlsbad, Calif.

l.

National Veterinary Service Laboratories, Ames, Iowa.

m.

StatView, version 2, SAS Institute Inc, Cary, NC.

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